US6793946B2 - Composition and method for lowering cholesterol - Google Patents

Composition and method for lowering cholesterol Download PDF

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US6793946B2
US6793946B2 US10/209,021 US20902102A US6793946B2 US 6793946 B2 US6793946 B2 US 6793946B2 US 20902102 A US20902102 A US 20902102A US 6793946 B2 US6793946 B2 US 6793946B2
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leaves
drymifolia
composition
cholesterol
milled
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US20040022883A1 (en
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Ernesto A. Brovelli
Julio Andres Vallejos
Haeri Roh-Schmidt
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Access Business Group International LLC
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Access Business Group International LLC
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Assigned to ACCESS BUSINESS GROUP INTERNATIONAL LLC reassignment ACCESS BUSINESS GROUP INTERNATIONAL LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: VALLEJOS, JULIO ANDRES, BROVELLI, ERNESTO A., ROH-SCHMIDT, HAERI
Priority to TW092120815A priority patent/TW200404560A/en
Priority to KR1020057001773A priority patent/KR20050026084A/en
Priority to AU2003257051A priority patent/AU2003257051A1/en
Priority to JP2004527681A priority patent/JP2005534714A/en
Priority to PCT/US2003/023885 priority patent/WO2004014298A2/en
Priority to CNA038220555A priority patent/CN1681523A/en
Publication of US20040022883A1 publication Critical patent/US20040022883A1/en
Priority to US10/855,301 priority patent/US20050208163A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F3/00—Tea; Tea substitutes; Preparations thereof
    • A23F3/06—Treating tea before extraction; Preparations produced thereby
    • A23F3/14—Tea preparations, e.g. using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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  • the present invention relates to compositions and methods for lowering cholesterol. More particularly, the present invention relates compositions and methods having avocado leaf of the species Persea americana var. drymifolia (hereinafter referred to as “ drymifolia ”) for use in lowering cholesterol levels in humans.
  • drymifolia avocado leaf of the species Persea americana var. drymifolia
  • Cholesterol is a soft, waxy substance found among the lipids in the bloodstream and in cells of the human body. Although cholesterol serves needed bodily functions, too high a level of cholesterol in the blood may be detrimental to a person's health because it increases the risk of cardiovascular disease. High cholesterol generally means that a person's total blood cholesterol level is more than 240 mg/dl or that a person's low density lipoprotein level is more than 160 mg/dl (see, Cleeman, James I., Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III), JAMA 285(19): 2486-2497 (2001)).
  • NCEP National Cholesterol Education Program
  • LDL low-density lipoproteins
  • HDL high density proteins
  • statins There are several classes of available lipid-regulating pharmacological interventions called statins.
  • statins One common statin is LIPITOR®. Although these agents have been proven safe in clinical trials, like any drug, they carry the risk for side-effects. Most notable of the side-effects is myopathy, which becomes evident as muscle pains and weakness.
  • the present invention is a composition containing avocado leaves from the variety drymifolia for reducing cholesterol levels.
  • the leaves are dehydrated and milled for consumption as a tea.
  • the leaves of drymifolia showed a comparable cholesterol lowering effect to LIPITOR® and a much greater cholesterol lowering effect than the fruit and the leaves of Persea nubigena var. guatamalensis cv. Nabal and Persea nubigena var. guatamalensis cv. Haas, which are avocados commonly grown in North America.
  • FIG. 1 is a graph displaying dose specific responses of Hepatocyte G2 cells to hand-crushed and milled leaves of drymifolia in comparison to LIPITOR® in cholesterol release.
  • FIG. 2 is a graph displaying dose specific responses of Hepatocyte G2 cells to milled leaves of drymifolia in comparison to milled leaves of Persea nubigena var. guatamalensis cv. Nabal and milled leaves of Persea nubigena var. guatamalensis cv. Haas in cholesterol release.
  • FIG. 3 is a graph displaying dose specific responses of Hepatocyte G2 cells to milled leaves of drymifolia in comparison to the fruit of Persea nubigena var. guatamalensis cv. Haas in cholesterol release.
  • the present invention comprises avocado leaves from the variety Persea americana var. drymifolia (“ drymifolia ”). This variety is available in Northern Argentina. The particular substance(s) in the leaves that causes cholesterol lowering is not yet understood. However, it is a commonly understood principle in the herbal industry that active ingredients in fresh plants/herbs generally decompose or diminish in effectiveness as the plant/herb dies or decays. Thus, to preserve the cholesterol reducing capacity of the drymifolia leaves for shipment from Argentina to the United States, the fresh drymifolia leaves are preferably air dried at average room temperatures of about 60° F. to about 80° F. This temperature range is maintained until the leaves are prepared for consumption. Surprisingly, this preconditioning step maintains the efficacy of the drymifolia leaves in lowering cholesterol.
  • the leaves are preferably hand-crushed.
  • the hand-crushed leaves showed better cholesterol lowering activity than the fruit and leaves of Persea nubigena var. guatamalensis cv. Nabal (“Nabal”) and the leaves of Persea nubigena var. guatamalensis cv. Haas (“Haas”).
  • dehydrated leaves are milled using a 2 mm screen to mill the leaves to an average particle size of about 28 microns. Even more preferably, the leaves may be milled twice using a 1 mm screen to provide an average particle size of about 9 microns.
  • the dehydrated leaves may be milled using a 1 mm screen, providing an average particle size of about 11 microns. Milling can be performed by a GlenMills hammer mill bench top unit (Mot. KM 80-60, Culatti Typ MFC). Milled drymifolia leaves showed greater cholesterol reduction than hand-crushed drymifolia leaves. It is contemplated that the milled and dehydrated leaves can be used in the form of tea, capsules, tablets, creams, gels, liquids, and foods. However, for exemplary purposes, preparation and consumption in the form of a tea is described.
  • the tea is prepared as a 1% extract by preparing 10 mg of hand-crushed drymifolia leaves or milled drymifolia leaves per 1 ml of water. In one embodiment, 100 mg of drymifolia leaves are placed in 10 ml of water to form a tea. The tea is boiled for about 1 minute to about 10 minutes. More preferably, the tea is boiled for about 3 minutes to about 7 minutes and, most preferably, for about 5 minutes. The water can be boiling after or upon immersing the drymifolia leaves in the water. It will be appreciated by those of ordinary skill in the art that the boiling times will vary depending upon the volume of the tea.
  • LIPITOR® is prepared in methanol/buffer to solubilize atorvastatin, the active ingredient in LIPITOR® (40 mg active/600 mg tablet). All test materials were applied to serum culture medium at varying doses.
  • DMEM Dulbecco's Modified Eagle Medium
  • DMEM Dulbecco's Modified Eagle Medium
  • the final concentration is about 0.2 mg/ml to about 0.4 mg/ml drymifolia leaves and, most preferably, about 0.4 mg/ml drymifolia leaves.
  • the culture media provides all necessary nutrients for cell maintenance including cholesterol synthesis.
  • Amounts of secreted cholesterol and cholesteryl ester were measured from acetate fed hepatocyte culture media using a fluorescent indicator, AMPLEX RED. As shown in FIG. 1, it is apparent that the effect of milled drymifolia leaves is comparable to that of LIPITOR®. The results also show that milled drymifolia leaves with an average particle size of about 11 microns had a more favorable result than that of hand-crushed drymifolia leaves when water extracts are made from each. The response of hepatocytes also indicated little or no toxicity associated with the doses treated when cell survival post treatment was assessed via MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide, Sigma, St. Louis, Mo.) reduction assay.
  • MTT 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide
  • FIG. 2 shows the effect of the milled drymifolia leaves to milled leaves of Haas and Nabal.
  • FIG. 2 shows that drymifolia leaves have a more favorable effect in decreasing cholesterol synthesis/secretion than that of Haas or Nabal.

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Abstract

The present invention relates to a composition comprising Persea american var. drymifolia for lowering cholesterol levels.

Description

FIELD OF THE INVENTION
The present invention relates to compositions and methods for lowering cholesterol. More particularly, the present invention relates compositions and methods having avocado leaf of the species Persea americana var. drymifolia (hereinafter referred to as “drymifolia”) for use in lowering cholesterol levels in humans.
BACKGROUND OF THE INVENTION
Cholesterol is a soft, waxy substance found among the lipids in the bloodstream and in cells of the human body. Although cholesterol serves needed bodily functions, too high a level of cholesterol in the blood may be detrimental to a person's health because it increases the risk of cardiovascular disease. High cholesterol generally means that a person's total blood cholesterol level is more than 240 mg/dl or that a person's low density lipoprotein level is more than 160 mg/dl (see, Cleeman, James I., Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III), JAMA 285(19): 2486-2497 (2001)). Approximately 41.3 million Americans have total blood cholesterol levels of 240mg/dL or higher (see, American Heart Association, Biostatistical Fact Sheet, www.americanheart.org (2002)). When blood cholesterol reaches these high levels, it can build up on artery walls because cholesterol does not dissolve in the blood. Rather, cholesterol has to be transported to and from the cells by special carriers called low-density lipoproteins (LDL) and high density proteins (HDL). HDL carries cholesterol away from your arteries and is, thus, considered “good cholesterol.” Too much LDL cholesterol can clog the arteries to your heart and is, thus, considered “bad cholesterol.” This condition, called atherosclerosis, increases the risk of blood clots, heart attack, and stroke.
Accordingly, there is considerable amount of interest in regulating cholesterol level in the body. There are several classes of available lipid-regulating pharmacological interventions called statins. One common statin is LIPITOR®. Although these agents have been proven safe in clinical trials, like any drug, they carry the risk for side-effects. Most notable of the side-effects is myopathy, which becomes evident as muscle pains and weakness. Furthermore, it is important to minimize the potential for adverse reactions or drug interactions when a patient is undergoing statin therapy (see, Davidson, Michael H., Treatment of the Elderly with 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors: Focus on Drug Interactions, J Cardiovasc Pharmacol Therapeut, 6(3): 219-229 (2001)). In its Adult Treatment Panel, the National Cholesterol Education Program recommends the use of plant derived substances in the adjuvant therapies for dyslipidemias (see Cleeman, supra). Accordingly, natural alternatives with effective and safe cholesterol reduction are desired.
SUMMARY OF THE INVENTION
The present invention is a composition containing avocado leaves from the variety drymifolia for reducing cholesterol levels. In one embodiment the leaves are dehydrated and milled for consumption as a tea. Surprisingly, the leaves of drymifolia showed a comparable cholesterol lowering effect to LIPITOR® and a much greater cholesterol lowering effect than the fruit and the leaves of Persea nubigena var. guatamalensis cv. Nabal and Persea nubigena var. guatamalensis cv. Haas, which are avocados commonly grown in North America.
These and other aspects and advantages of the present invention will be better understood by reference to the drawings and the detailed description of the preferred embodiment. It is noted that, unless otherwise stated, all percentages given in this specification and the appended claims refer to percentages by weight.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph displaying dose specific responses of Hepatocyte G2 cells to hand-crushed and milled leaves of drymifolia in comparison to LIPITOR® in cholesterol release.
FIG. 2 is a graph displaying dose specific responses of Hepatocyte G2 cells to milled leaves of drymifolia in comparison to milled leaves of Persea nubigena var. guatamalensis cv. Nabal and milled leaves of Persea nubigena var. guatamalensis cv. Haas in cholesterol release.
FIG. 3 is a graph displaying dose specific responses of Hepatocyte G2 cells to milled leaves of drymifolia in comparison to the fruit of Persea nubigena var. guatamalensis cv. Haas in cholesterol release.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention comprises avocado leaves from the variety Persea americana var. drymifolia (“drymifolia”). This variety is available in Northern Argentina. The particular substance(s) in the leaves that causes cholesterol lowering is not yet understood. However, it is a commonly understood principle in the herbal industry that active ingredients in fresh plants/herbs generally decompose or diminish in effectiveness as the plant/herb dies or decays. Thus, to preserve the cholesterol reducing capacity of the drymifolia leaves for shipment from Argentina to the United States, the fresh drymifolia leaves are preferably air dried at average room temperatures of about 60° F. to about 80° F. This temperature range is maintained until the leaves are prepared for consumption. Surprisingly, this preconditioning step maintains the efficacy of the drymifolia leaves in lowering cholesterol.
Once dehydrated, the leaves are preferably hand-crushed. The hand-crushed leaves showed better cholesterol lowering activity than the fruit and leaves of Persea nubigena var. guatamalensis cv. Nabal (“Nabal”) and the leaves of Persea nubigena var. guatamalensis cv. Haas (“Haas”). More preferably, dehydrated leaves are milled using a 2 mm screen to mill the leaves to an average particle size of about 28 microns. Even more preferably, the leaves may be milled twice using a 1 mm screen to provide an average particle size of about 9 microns. Most preferably, the dehydrated leaves may be milled using a 1 mm screen, providing an average particle size of about 11 microns. Milling can be performed by a GlenMills hammer mill bench top unit (Mot. KM 80-60, Culatti Typ MFC). Milled drymifolia leaves showed greater cholesterol reduction than hand-crushed drymifolia leaves. It is contemplated that the milled and dehydrated leaves can be used in the form of tea, capsules, tablets, creams, gels, liquids, and foods. However, for exemplary purposes, preparation and consumption in the form of a tea is described.
The tea is prepared as a 1% extract by preparing 10 mg of hand-crushed drymifolia leaves or milled drymifolia leaves per 1 ml of water. In one embodiment, 100 mg of drymifolia leaves are placed in 10 ml of water to form a tea. The tea is boiled for about 1 minute to about 10 minutes. More preferably, the tea is boiled for about 3 minutes to about 7 minutes and, most preferably, for about 5 minutes. The water can be boiling after or upon immersing the drymifolia leaves in the water. It will be appreciated by those of ordinary skill in the art that the boiling times will vary depending upon the volume of the tea.
Drymifolia Comparison Studies
To test the efficacy of the drymifolia tea containing milled drymifolia leaves having an average particle size of about 11 microns, comparison studies were performed with tea containing hand-crushed drymifolia leaves, LIPITOR®, Haas fruit, and milled leaves of Haas and Nabal having an average particle size of about 11 microns. LIPITOR® is prepared in methanol/buffer to solubilize atorvastatin, the active ingredient in LIPITOR® (40 mg active/600 mg tablet). All test materials were applied to serum culture medium at varying doses.
Tea containing drymifolia leaves having an average particle size of about 11 microns was added to Dulbecco's Modified Eagle Medium (DMEM, Catalogue #11965, In Vitrogen Corporation, 1600 Faraday Avenue, P.O. Box 6482, Carlsbad, Calif. 92008) to a final concentration of about 0.1 mg/ml to about 0.5 mg/ml drymifolia leaves via serial dilution (or 0.01% to 0.05% drymifolia leaves). More preferably, the final concentration is about 0.2 mg/ml to about 0.4 mg/ml drymifolia leaves and, most preferably, about 0.4 mg/ml drymifolia leaves. The culture media provides all necessary nutrients for cell maintenance including cholesterol synthesis.
Molarity
Components of Culture Medium (mM)
INORGANIC SALTS:
Calcium Chloride (CaCl2) (anhyd.) 1.80
Ferric Nitrate (Fe(NO3)3-9H2O) 0.000248
Potassium Chloride (KCl) 5.30
Magnesium Sulfate (MgSO4) 0.813
Sodium Chloride (NaCl) 110.34
Sodium Bicarbonate (NaHCO3) 44.10
Sodium Phosphate (NaH2PO4-H2O) 0.906
OTHER COMPONENTS:
D-Glucose 25.00
Phenol red 0.0346
AMINO ACIDS:
L-Arginine-HCl 0.398
L-Cystine 2HCl 0.200
L-Glutamine 4.00
Glycine 0.399
L-Histidine HCl-H2O 0.20
L-Isoleucine 0.802
L-Leucine 0.802
L-Lysine-HCl 0.798
L-Methionine 0.201
L-Phenylalanine 0.400
L-Serine 0.400
L-Threonine 0.078
L-Tryptophan 0.078
L-Tyrosine 2Na 2H2O 0.398
L-Valine 0.803
VITAMINS:
D-Ca pantothenate 0.0083
Choline Chloride 0.0285
Folic Acid 0.00906
i-Inositol 0.04
Niacinamide 0.0328
Pyridoxine HCl 0.0196
Riboflavin 0.00106
Thiamine HCl 0.0118
Amounts of secreted cholesterol and cholesteryl ester were measured from acetate fed hepatocyte culture media using a fluorescent indicator, AMPLEX RED. As shown in FIG. 1, it is apparent that the effect of milled drymifolia leaves is comparable to that of LIPITOR®. The results also show that milled drymifolia leaves with an average particle size of about 11 microns had a more favorable result than that of hand-crushed drymifolia leaves when water extracts are made from each. The response of hepatocytes also indicated little or no toxicity associated with the doses treated when cell survival post treatment was assessed via MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide, Sigma, St. Louis, Mo.) reduction assay.
FIG. 2 shows the effect of the milled drymifolia leaves to milled leaves of Haas and Nabal. FIG. 2 shows that drymifolia leaves have a more favorable effect in decreasing cholesterol synthesis/secretion than that of Haas or Nabal.
As shown in FIG. 3, when the cholesterol inhibitory effect of Haas fruit is assessed in comparison to that of the milled drymifolia leaves, the milled leaves again exerted much more favorable response than the fruit.
While the above describes what are presently believed to be the preferred embodiments of the invention, those skilled in the art will realize that changes and modifications may be made thereto without departing from the spirit of the invention. It is intended to claim all such changes and modifications that fall within the true scope of the invention.

Claims (7)

What is claimed:
1. A composition for lowering cholesterol levels comprising about 0.01% to about 0.05% of Persea americana var. drymifolia leaves by total weight of said composition wherein said drymifolia leaves have an average particle size of about 9 microns to about 28 microns.
2. The composition of claim 1 wherein said composition is a beverage containing drymifolia leaves.
3. The composition of claim 1 wherein said composition is a dietary supplement.
4. The composition of claim 1 wherein said composition lowers total blood cholesterol levels by about 10% to about 40%.
5. The composition of claim 1 wherein said Persea americana var. drymifolia leaves have an average particle size of about 11 microns.
6. The composition of claim 1 wherein said Persea americana var. drymifolia leaves are present in the amount of about 0.04% by total weight of said composition.
7. A tea for lowering cholesterol levels comprising about 0.1 mg/ml to about 0.5 mg/ml of Persea americana var. drymifolia leaves.
US10/209,021 2002-07-31 2002-07-31 Composition and method for lowering cholesterol Expired - Fee Related US6793946B2 (en)

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US10/209,021 US6793946B2 (en) 2002-07-31 2002-07-31 Composition and method for lowering cholesterol
TW092120815A TW200404560A (en) 2002-07-31 2003-07-30 Composition and method for lowering cholesterol
JP2004527681A JP2005534714A (en) 2002-07-31 2003-07-31 Compositions and methods for lowering cholesterol
AU2003257051A AU2003257051A1 (en) 2002-07-31 2003-07-31 Composition and method for lowering cholesterol
KR1020057001773A KR20050026084A (en) 2002-07-31 2003-07-31 Composition and method for lowering cholesterol
PCT/US2003/023885 WO2004014298A2 (en) 2002-07-31 2003-07-31 Composition and method for lowering cholesterol
CNA038220555A CN1681523A (en) 2002-07-31 2003-07-31 Composition and method for lowering cholesterol
US10/855,301 US20050208163A1 (en) 2002-07-31 2004-05-27 Compositions containing avocado leaf extract for lowering cholesterol levels

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WO2005100592A1 (en) * 2004-04-13 2005-10-27 Rush University Medical Center A pharmacologic method of lowering cholesterol production
EP1750695B1 (en) * 2004-05-28 2010-03-31 Laboratoires Expanscience Use of furan alkyls for preparing a drug for treating obesity and cosmetically treating overweight
FR2870742B1 (en) 2004-05-28 2008-03-14 Expanscience Laboratoires Sa USE OF ALKYL FURANS FOR THE PREPARATION OF A MEDICAMENT FOR THE TREATMENT OF DIABETES, OBESITY AND FOR THE COSMETIC TREATMENT OF CELLULITE AND OVERLOAD
TWI605822B (en) * 2015-04-09 2017-11-21 高雄醫學大學 Uses of an avocado extract, avocadenol b, and (2r,4r)-1,2,4-trihydroxyheptadec-16-yne, and health food containing the avocado extract
WO2017063150A1 (en) * 2015-10-14 2017-04-20 高雄医学大学 Application of avocado extract, avocadenol b, and (2r,4r)-1,2,4-trihydroxy heptadec-16-yne, and health food comprising avocado extract
WO2018122218A1 (en) 2016-12-29 2018-07-05 Koninklijke Philips N.V. Coffee brewing
CN112425668A (en) * 2020-09-28 2021-03-02 广西南亚热带农业科学研究所 Processing method of avocado leaf green tea

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